Ancient DNA is revealing new insights into the genetic relationship between Pleistocene hominins and modern humans. Nuclear DNA indicated Neanderthals as a sister group of Denisovans after diverging from modern humans. However, the closer affinity of the Neanderthal mitochondrial DNA (mtDNA) to modern humans than Denisovans has recently been suggested as the result of gene flow from an African source into Neanderthals before 100,000 years ago. Here we report the complete mtDNA of an archaic femur from the Hohlenstein–Stadel (HST) cave in southwestern Germany. HST carries the deepest divergent mtDNA lineage that splits from other Neanderthals ∼270,000 years ago, providing a lower boundary for the time of the putative mtDNAintrogression event. We demonstrate that a complete Neanderthal mtDNA replacement is feasible over this time interval even with minimal homininintrogression. The highly divergent HST branch is indicative of greater mtDNA diversity during the Middle Pleistocene than in later periods.

Acknowledgements

We are grateful for comments from Ilan Gronau, Adam Powell, Wolfgang Haak, Maria Spyrou and Alvise Barbieri. We thank Frido Welker and Matthew Collins for ZooMS analyses, Alexander Peltzer and Gabriel Renaud for support in running EAGER and schmutzi, respectively, Mannis van Oven for access to the updated version of the RNRS reference and Annette Günzel for graphical support. C.P. and J.K. were supported by the Baden Württemberg Foundation and the Max Planck Society.